Process for producing colorless sterile invert sugar solution



United States Patent PROCESS FOR PRODUCING COLORLESS STERILE INVERTSUGAR SOLUTION Lawrence J. Heidt, Arlington, Mass., assignor, by mesncassignments, to Sugar Research Foundation, Inc., New York, N. Y., acorporation of New York No Drawing. Application July 31, 1951, SerialNo. 239,635

9 Claims. (Cl. 127-41) This application is a continuation-in-part of thecopending application, Serial No. 114,455, filed September 7, 1949,which was a continuation-in-part of the application, Serial No. 758,995,filed July 3, 1947.

This invention relates to invert sugar and more particularly to sterilestable solutions of invert sugar and their preparation by the inversionof sucrose.

An object of this invention is to produce invert sugar which contains aminimum quantity of the decomposition product of levulose.

Another object of this invention is to provide a sterile water whitesolution of invert sugar which is substantially free fromS-hydroxymethylfurfural.

Another object of this invention is to sterilize by heat, solutions ofinvert sugar which are suitable for parenteral administration withoutthe production of detectable quantities of S-hydroxymethylfurfural asdetermined by standard color tests.

Sterile solutions of invert sugar have been heretofore proposed asalternatives for solutions of glucose employed therapeutically forparenteral administration,

However, such solutions of invert sugar contain contaminants, asevidenced by a yellow or brownish cast of the solution. Such solutionsare relatively unstable, producing decomposition products over a periodof time which further contaminate the solution of invert sugar.

During sterilization by heat, solutions of invert sugar are especiallyprone to further decomposition. Since untoward reactions sometimesresult from the presence of these contaminants, particularly if a markedquantity is present, many such Solutions are not utilizedtherapeutieally.

In accordance with this invention, invert sugar is produced which issubstantially free of undesirable decomposition products. Moreparticularly, sterile solutions of invert sugar are produced which arewater white in visible light to the naked eye when viewed through adepth of 4 inches and are stable over prolonged periods, such as atleast one year at room temperature. Solu tions produced in accordancewith this invention contain an amount by weight ofS-hydroxymethylfurfural not more than 0.2%, and preferably not more than0.1%, of the heXose in the solution. Even when such solutions containthis small amount of s-hydroxymethylfurfural, the amount of sucrose insuch solutions is less than 2% of the total sugars present.

ice

The development of color by sugar and its solutions is usually referredto as browning. Along with this color there is always present5-hydroxymethylfurfural commonly called hydroxymethylfurfural. 5hydroxymethylfurfural has also been found to be present when browning isabsent, but the amount that can be present without browning is of theorder of 0.2% or less by weight of the total sugar.S-hydroxymethylfurfural is itself colorless, but its concentration andthe intensity of browning are roughly directly proportional to eachother at a given concentration of hexose. The factor of proportionality,however, varies with the pH of the material and to a smaller extent withthe way in which the color has been produced. In particular, I havefound that the ratio of the concentration of S-hydroxymethylfurfural tobrowning is larger at pH 3 than at pH 4 and continues to decrease as thepH increases.

In invert sugar solution, browning is produced mainly by thedecomposition of levulose since its production by dextrose is very muchslower.

The production of colorless solutions of invert sugar by thesubstantially complete inversion of sucrose by acid I have foundinvolves the precise control of the certain specific variables. Thesevariables are the pH of the solution, the temperature at which theinversion is carried out, the initial concentration of sucrose, and theamount of levulose oxidized by such substances as the oxygen present inthe solution.

The sterile water white solutions of this invention are produced byinitially hydrolyzing aqueous solutions of sucrose under controlledconditions to convert 98 to 99.9% of the sucrose to invert sugar andsimultaneously or thereafter sterilizing by heat the hydrolyzed solutionalso under controlled conditions. The inversion of the sucrose iseflected by hydrolyzing 98 to 99.9% of the sucrose in an aqueuossolution having a pH of 0.0 to 4.0 in the substantial absence of anoxidizing agent and at a temperature within the range of 0 to C. Theconcentration of the sucrose prior to hydrolysis should be not more than30% when the hydrolysis is efiected at a'pH above 3 and not more than10% when the hydrolysis is eliected at a pH above 3.5. When it isnecessary to efliect sterilization after hydrolysis, the hydrolyzedsolution is sterilized by heat under such conditions that the resultingsterilized solution has a pH within the range of 2.5 to 4.9.

The steps of hydrolyzing and sterilization may be conducted in sequenceor simultaneously. Consequently, both in this description and in theclaims, this invention includes within its scope the practice of bothsteps whether conducted in sequence or simultaneously.

The inversion and sterilization are eliected in the substantial absenceof an oxidizing agent in the solution. By oxidizing agent is meant anysubstance at a concentration which will oxidize sugar in an aqueoussolution. For example, nitric acid would be such a substance if presentat a concentration of greater than 0.1 N. Oxygen in water in equilibriumwith the atmosphere is also such an oxidizing agent when the pH of thewater is above about 2.3, but is not such an oxidizing agent up to atleast a pi-l of 4.9 when the major portion of the oxygen is re- .aovcd.Throughout the description and claims, the removal of the major portionof oxygen from the solution means the removal of at least 50% of theoxygen soluble in the solution at room temperature under atmosphericconditions.

If the hydrolysis is conducted within the range of 2.3 to 4.0, the majorportion of the oxygen should be removed therefrom by any suitable means,such as physical evacuation of the oxygen by reducing the pressure. Oneconvenient method of removal of the major portion of the oxygen is toadjust the solution to a pH of 2.0 or lower with a non-oxidizing acid,such as hydrochloric acid, followed by addition of a non-toxic salt ofcarbonic acid, such as sodium bicarbonate or calcium carbonate until thepH of the solution is within the range of 2.3 and 3.0 whereby the oxygenin the solution is replaced by carbon dioxide. Another convenient methodof removing the major portion of the oxygen is to add a sufficientamount of a non-toxic salt of carbonic acid to an acid solution untilthe pH of the solution is between 2.3 and 4.0.

The hydrolysis is effected in an acid solution produced by anon-oxidizing acid, such as mineral acids, for example, hydrochloricacid or sulfuric acid; organic acids, for example, acetic acid, lacticacid or tartaric acid; or acidic resins, for example, cation exchangeresins; or any combination of such acids. It is essential that, when thesterile water white solutions of this invention are intended forparenteral use, the acid or any reaction product thereof remaining inthe solutions after hydrolysis and sterilization be non-toxic. When aninsoluble acid, such as a cation exchange resin, is employed foreffecting hydrolysis, it is removed prior to sterilization and asoluble, non-oxidizing acid is employed for obtaining the required pHfor the sterilization step.

When the pH of the hydrolyzed solution of invert sugar is such that thesolution after sterilization by heat is not within the range of 2.5 to4.9, it is adjusted to the required pH to produce such a solution. Thisadjustment is preferably made by the addition of a non-toxic salt ofcarbonic acid to the solution of invert sugar when the pH is too low, orby the addition of a soluble nonoxidizing, non-toxic acid followed bythe addition of a sutficient amount of a non-toxic salt of carbonic acidto the solution of invert sugar when the pH is too high, as, forexample, when the hydrolyzed solution results from the use of an anionexchanger.

The hydrolyzed solution having a pH such that when sterilized it willhave a pH Within the range of 2.5 and 4.9 is sterilized by heat by anyconvenient means, such as by autoclaving. The sterilized solution isthen cooled to room temperature and can be maintained in a water whitecondition for a period of at least a year.

The methods of this invention are especially suited to the preparationof sterile solutions of invert sugar for parenteral use. Solutionsproduced by these methods differ from sterile solutions of invert sugarheretofore produced in that the sterile solutions of this invention arecolorless and contain an amount by weight of S-hydroxymethylfurfural notmore than 0.2%, and preferably not more than 0.1%, of the hexose in thesolution. Moreover, the sterile, colorless solutions of this inventionat a pH of 4.0 or greater do not produce untoward reactions whenadministered parenterally.

The presence of the carbon dioxide in the sterile solution resultingfrom the neutralization with a non-toxic salt of carbonic acid effects amarked prolongation of the stability of the solution of invert sugar.The carbon dioxide generated in the neutralization step replaces theoxygen contained therein, and the absence of the major portion of oxygenin the solution lessens to a marked degree the tendency of the invertsugar to decompose.

The inversion of between 98 and 99.9% of the sucrose can be controlledadequately without the production of color if the conditions chosen forthe inversion are such as to require at least fifteen minutes to invert99% of the sucrose. This percent of the sucrose can be inverted infifteen minutes, at a pH of 0.1 at 50 C. or at a pH of 2.3 at 100 C.These pH values are for a 2% sucrose solution; they would be higher athigher initial concentrations of sucrose or at higher temperatures.

If the solutions of invert sugar are required for parenteral use,non-pyrogenic material should be employed, the solutions should befiltered before the sucrose is inverted, and the invert sugar solutionsshould be sterilized. If non-pyrogenic materials are used, anonpyrogenic sterile solution of invert sugar results. For

therapeutic use, certain acids, such as hydrochloric acid, and certainalkali metal bicarbonates, such as sodium bicarbonate, are moreadvantageously used for the inversion and neutralization respectively.Solutions of invert sugar of this invention, particularly the saturatedsolutions of invert sugar, may be employed for othe uses, such asingredients of beverages, candies and ice creams, and for these otheruses, a water soluble nonoxidizing acid other than hydrochloric acidwhich produces a solution having a pH within the range of 0.0 to 4.0,such as sulfuric acid, phosphoric acid, tartaric acid, citric acid,fumaric acid, maleic acid, malic acid and malonic acid may be employed.Some of these nonoxidizing acids may be objectionable for certain uses.Hydrochloric acid is particularly adapted for certain uses, such as forsterile invert sugar solution for parenteral administration.

When a sterile water white invert sugar solution having a final pH of2.5 to 4.0 is desired, the inversion of up to 99.9% of the sucrose andthe sterilization by heat may also be carried out simultaneously byautoclaving at fifteen pounds steam pressure for twenty to thirtyminutes or at seven pounds steam pressure for thirty to sixty minutes.

The pH of all solutions is determined at room tern-- perature by meansof a properly calibrated pH meter em ploying a glass electrode.

All percentages of the components of the solution-,- equal 100 times thegrams of the component per 100 ml. of the solution at room temperature.

The amount of 5-hydroxymethylfurfural in sugar solutions produced by theinversion of sucrose may be determined by means of a polarographutilizing the information disclosed, and the references cited, in thepaper by S. N. Cantor and Q. P. Peniston in the Journal of AmericanChemical Society, 62 21 13 (1940). I have found that the presence ofinvert sugar and sucrose does not interfere with the determinaiton ofthe concentration of S-hydroxymethylfurfural by means of a polarograph.

Cantor et al. found that the concentration of S-hydroxymethylfurfural inwater solution was directly proportional to the diffusion current (waveheight) produced by it. The proportionality factor was found to beindependent of the pH of the solution as long as the concentration ofS-hydroxymethylfurfural did not exceed 1 10 molar as disclosed by Figure4 of their article. In solutions containing a higher concentration of 5-hydroxymethlyfurfural, the concentration of this substance can bereduced to 1X10 molar or less by appropriate dilution. The amount of5-hydroxymethylfurfural in the original solution (raw solution) can thenbe calculated by multiplication of the observed concentration by theappropriate factor.

The application of this analytical method to the solutions of invertsugar produced by the methods of this invention shows that even thecolorless sterile solutions contain measurable concentrations ofS-hydroxymethylfurfural but that the concentration of this substance didnot exceed 0.2% of the hexoses present when color was absent and greatlyexceeded this amount in the colored solutions. These facts are revealedin the following ferences between the values before and aftersterilization table: in the last SIX columns correspond, except at pH4.6,

TABLE I Polarographic determination of the concentration ofS-hydmxymethylfurfural in certain solutions Cone. of 6- Percent Out inthe pH of Polarohydroxy- Hexose con- Sarnple No. Raw Solution cone. ofSolution Galv. gram El in id. in methylfurverted to the raw AnalyzedSens. No. Volts p amps. fural in moles 5-hydroxysolution per liter ofthe methylfur- Raw Solution fural l 6. 9 l 1 none 1 6.9 10 2 none 1 6. 910 3 none 1 6. 8 10 4 none Levnlose. Overland Pure Honey tie 6. 8 2 5none Saturated Invert Sugar solution at pH he 6. 9 2 6 l.21 0.021.

2.8 made by process in accordance with Example 3, infra. Same $63 7.0 l7 1.22 .29 1.05 10 0.018. Same %X%3 7.0 1 7 1.24 .052 0.929X10' 0.016.10% Invert Sugar Solution in accordti 6. 7 10 8 1.24 2. 8 0.874 10-0.15.

arllice with Example 2, sterilized at D 3.1. Same a 6.8 10 9 l. 23 3.00.93X10- 0.16. Same M0 6.8 10 9 1. 24 .57 0.874X10- 0.15. Same butsterilized at pH 3.7-. )2 6. 7 5 10 l. 22 99 0.306X10- 0.053. Same butsterilized at pH 4.2-. la 6. 7 10 11 1. 23 8 0.248Xl0' 0.043. Same butsterilized at pH 4.6 $2 6. 7 5 12 l. .47 0.145X10-L. 0.025. Same exceptsterilized at pH 6.1 and la 7.0 5 l3 greater brown. 7 than 1% 5%Dextrose sterilized at pH 3.8 by 2 6.7 5 14 1.23 .14 0.0436Xl0-L 0 015.

Abbott Laboratories Inc.

The sucrose employed in the measurements was Revere extra finegranulated cane sugar, purchased in a sealed paper bag from a localgrocer.

The dextrose was purchased in a sealed paper carton and was Squibbs U.S. P. XII anhydrous material, equal or superior to what is commonlyknown as C. P.

The levulose was C. P. special crystals of D-fructose and was part ofPhanstiehls lot No. 1378 The Overland Pure Honey was purchased in asealed glass jar from a local grocer.

A more comprehensive understanding of this invention is obtained byreference to the following examples:

EXAMPLE 1 Twelve i002 ml. of 12 N hydrochloric acid (C. P. reagent acid,sp. gr. about 1.19) are thoroughly mixed with 500 ml. Water. Ninety-fivegrams of sucrose are dissolved in the acid solution and inverted bykeeping the solution at 50il C. for 60:3 minutes. The solution isneutralized by mixing in slowly and thoroughly a volume of 0.3 M sodiumbicarbonate until the pH is within the range 2.5 to 4.9. The inversionand neutralization are carried out in a suitable bottle which isadequately vented and in which the solution can be sterilized by moistheat at 250 F. for 20 to minutes. After sterilization, the bottle ishermetically sealed as soon as the autoclave is opened. The autoclave isopened as soon as it has cooled to 180 F.

Data showing that the invert sugar in the aqueous saline solutionsprepared by the above example, which is an isotonic solution, is notaffected measurably during sterilization for 20 to 30 minutes at 250 F.when the pH of the solution introduced into the autoclave is within therange of 2.5 to 4.9 are given in the following table. The solutionsremained water white. The data are for aqueous solutions containing 10percent invert sugar and 0.85 percent sodium chloride. More than 98percent of the sucrose had been inverted. The difto the 3 to 4 percentloss of water during sterilization.

pH SHS KA Optical Activity before after before after before after beforeafter 2.9 3. 0 21. 8 22. 5 25. 9 26. 4 1. 65 l. 71 3. 4 3. 5 21. 7 22. 425. 7 26. 4 1. 65 l. 71 4. 2 4. 3 22. 0 22. 6 25. 6 26. 5 1. 66 l. 68 4.6 4. 8 21.7 22. 3 25.8 25. 9 l. 62 1. 61

The caption SHS in the above table indicates that the values wereobtained by the Shafier, Hartman and Soinogyi method [Journal ofBiological Chemistry 45:377 (1920-21) ::695 (1933)] of estimatingreducing sugars, and was carried out on 5 ml. aliquots of solutionsproduced by diluting the original sugar solutions by a factor of 400.One unit of the entries in the table under the designation SHS isequivalent to 5.2 10* grams of invert sugar. Accordingly, about 98% ofthe sucrose was indicated as inverted. Under the designation KA in thetable are given the values of the estimated aldose content which weredetermined in accordance with the method of G. M. Kline and S. F. Acree[U. S. Bureau of Standards Journal of Research 5:1063 (1930)]. Thesevalues were obtained on 5 ml. samples of the original solutions. Oneunit of the entries in the table under KA is equivalent to 5X10- mole ofglucose. Accordingly, 53 instead of 50% of invert sugar might beconsidered glucose, but the 3% difference may be attributed to theuncertainty of the method as used in the presence of levulose. Theoptical activities given in the table were the observed values for the Dlines of sodium passing through one decimeter of solution at 2711' C.The sucrose inverted on this basis is 100% within the limits of error ofi0.01 in the polarimeter readings and :1 C. The uncertainty in thetemperature introduces the larger error because of the large change withtemperature in the optical activity of the levulose in water solution.

The solutions become discolored during sterilization when the pH at thebeginning of sterilization is outside the range of 2.5 to 4.9, and thefarther outside this pH range the more intense is the color. The tableshows that even in the solution at pH 4.6, which remained water whiteduring sterilization, a measurable loss in optical activity occurred.

The stability of invert sugar in water solution is increased by theremoval of the oxygen by the carbon dioxide produced during theneutralization with bicarbonate. The use of bicarbonate also insuresthat invert sugar is not lost during the neutralization by the momentarylocal production of pH values of 9 or over as might occur when strongalkaline solutions are used for the neutralization.

The inversion of the sucrose in 0.28 N HCl can be made over 98 percentcomplete by heating at 60 C. for 15 minutes, or at 57 C. for 20 minutesor at some other temperature for the proper time.

The sucrose solutions were brought quickly to the temperature at whichmost of the inversion was to take place by swirling them for a fewminutes in boiling water. This preheating and its dangers may beeliminated, however, if the solution is allowed to remain someadditional time in the constant temperature bath.

EXAMPLE 2 Ten percent colorless sterile invert sugar solutions wereprepared with 0.01 mole of sodium chloride per liter of solution. The pHrange covered was 2.5 to 4.9. The concentrat'ion of sodium chloride(0.01 M) in the final sterile sugar solutions was well below the valueof 0.04 M which in saline content is equivalent to the addition of purewater to blood.

The procedure consisted of inverting the sucrose in 0.024 N hydrochloricacid at 80 C. for 25 minutes, then neutralizing the solution with 0.03molar sodium bicarbonate until the desired pH was obtained. Thesolutions were then sterilized in an autoclave at 15 lbs. steam pressure(250 F.) for 20 to 30 minutes.

The buffer capacity of the sterile water white solutions at pH 4.0 andgreater which were prepared in accordance with Example 2 is less thanthat of the sterile dextrose solutions commercially available. It takesonly one-third as much alkali to bring to pH 7.4, the solutions of thisinvention having a pH of 4.7 as is required to bring to pH 7.4, the pHof the commercially available 10% dextrose solutions having a pH of 4.7.Accordingly, colorless sterile invert sugar solutions in isotonic sodiumchloride as described in Example 1 and in the equivalent of distilledwater which contain no butter are less acid at pH 4.7 than thecommercial dextrose solutions (pH 3.8 to 4.0) sold for intravenous use.

EXAMPLE 3 100 ml. of water is saturated with 500 g. of sucrose at roomtemperature. The mixture is stirred vigorously while 10 ml. of 1.2 Nhydrochloric acid are slowly added to it. This is followed by theaddition of enough (about 75 ml.) 0.15 M aqueous solution of sodiumbicarbonate to bring the pH between 2.5 and 3.0 (a pH of 2.8:01 isbest). The mixture is placed in a vented flask in an autoclave andheated at 15 lbs. steam pressure between twenty and thirty minutes. Thefinal product of this example is saturated aqueous solution of invertsugar.

This procedure inverts between 99 and 99.9% of the sucrose andsterilizes the solution without producing color (browning) or sutficient5-hydroxymethylfurfural to give a positive chemical test for it.

The saturated aqueous solution of invert sugar resulting from thepractice of the procedure of this example contains less than 1% byweight of sucrose and does not give a positive chemical test for5-hydroxymethylfurfural by the analytical research methods recommendedfor its detection, and shows less than 0.02% 5-1ydroxymethylfurfural byweight of the total sugar as measured with the aid of polarograph. Thesolution is also sterile and may be used if desired as an artificialhoney. The pH of native honey is 3.7:01. The pH of the above solution ofinvert sugar (artificial honey) may be brought to this pH by theaddition of sodium bicarbonate. If the artificial honey is used to cutnative honey, it is best to bring the pH of the native honey high enough(above 3.7) by the addition of sodium bicarbonate so that the pH of thecut honey is at 3.7:01 after mixing in the artificial honey.

The pH values cited above were obtained by means of a pH meter employinga glass electrode and a calomel half cell. The bulb of the glasselectrode was immersed in the sugar solution and the calomel half cellwas connected to the solution by a bridge of a saturated aqueoussolution of potassium chloride. The scale of the pH meter was calibratedby means of buffer tablets in each case dissolved in the specifiedvolume of water.

EXAMPLE 1 This example is directed to the preparation of a liter ofsolution of sterile water white invert sugar having a final,concentration of about 10 percent invert sugar, 0.07 percent sodiumchloride. The final pH is between 4.0 and 4.8 and the buffer capacity isbelow that of commercial sterile dextrose solutions for intravenousinjection.

The procedure used to produce the sterile water white solutions ofinvert sugar was as follows:

To a one liter sterilization bottle, 500 ml. water and 10 ml. of 1.2 HClwere added and mixed thoroughly. T this solution of hydrochloric acid,95 12 g. sucrose was added and the contents of the bottle werethoroughly mixed until the sucrose had dissolved completely.

The bottle was capped with a rubber bushing into which was inserted astainless steel cap which could serve as a vent or a seal. It wasessential to vent the bottle until the contents were sterilized, inorder that gases could escape from the bottle. The bottle and contentswere brought to 80i1 C. and held at this temperature for 25:2 minutes.The bottle and contents could be brought to 80 C. within minutes byswirling the bottle in a rapidly boiling water bath. Then, the bottlewas transferred at once to a bath at 80i1 C. for the required time. Ifthe bottle was transferred from room temperature to the bath at 80 C.,it took about 15 minutes for the bottle and contents to reach 80 C.Moreover, the bath temperature might drop several degrees when itreceived the cold bottle, and when this occurred, more than 15 minuteswere required to bring the material to 80 C.

The bottle was removed from the 80 C. bath to room temperature. Withintwo minutes, the cap and rubber bushing were removed from the hotbottle, the bottle was swirled, and while the contents were rotatingrapidly, the proper amount (380 to 400 ml.) of 0.03 M bicarbonate wascautiously added to bring the pH to 4.4:03.

The proper amount of 0.003 M bicarbonate was determined in the firstexperiments by adding initially about 375 ml. of the bicarbonate,mixing, and then determining the pH. If the pH was less than 4.3, thebicarbonate solution was added in lots of ml.; after each addition thebicarbonate solution added was mixed into the solution while the cap wasremoved from the bottle. The pH was then determined. This procedure wasrepeated until the pH reached 4.4:01. Thereafter, the proper amount of0.03 M bicarbonate to give a pH of 4.4103 could be added in oneoperation.

After the rubber bushing and cap were replaced on the sterilizationbottle and the bottle was adequately vented, the bottle and contentswere placed in an autoclave and sterilized at lbs. steam pressure (250F.) for to minutes. It should take no more than 8:3 minutes to bring thesteam pressure in the autoclave up to 15 lbs. After sterilization, thesteam was shut off and when the pressure had dropped to less than 2lbs., the door-of the autoclave was opened and the bottle caps werepushed down to seal the vents.

The solutions are colored (browned or caramelized) if the final pH isbelow 2.3 or above 5.1 when sterilized by heat, or if heated much longerthan the time required for sterilization or if more than 99.9 percent ofthe sucrose has been inverted or at any time strong acid, for example, 6N hydrochloric acid, or strong alkali, for example, 6 N sodiumhydroxide, has contacted the sugar solution for several minutesespecially at temperatures above 30 C. The solutions are pyrogen freeonly if pyrogen free materials are used with appropriate care. C. W.Walters, Surgery, Gynecology and Obstetrics 63, 643 (1936) gives amethodand the equipment necessary to prepare pyrogen free solutions forparenteral use.

The materials and equipment used. in the preparaiton of the sterilewater white solutions described in this example are as follows:

White granulated sucrose was purchased from a grocery, the paper bagcontainer was sealed at the refinery and the seal had not been broken.

The hydrochloric acid was C. P. reagent acid, sp. gr. about 1.19. Thesolution was diluted ten fold to make it 1.2 N HCl, e. g., 10 ml. of theacid was diluted to 100 ml. of solution.

The sodium bicarbonate was analytical reagent quality.

The water was freshly distilled from a clean still and condensingsystem.

The sugar solutions were made up, inverted, and sterilized in commercialsterilizationbottles of one liter capacity. Selected Fenwal bottles,rubber bushings, and stainless steel stoppers which provided ventingwere satisfactory. The equipment was carefully selected to insure properventing and an airtight seal when closed.

A platform balance sufiiced for weighting the sucrose and sodiumbicarbonate. v

A one day supply of 1.2 N HCl and 0.03 M bicarbonate (25g. NaHCOa perliter) solutions was made up at the beginning of each day. Thepreparation of each liter of sterile invert sugar solution required 10ml. of 1.2 N HCl and about 400 ml. of 0.03 M bicarbonate. Theconcentrations were within percent of the above values, but it wasimportant that. they were uniform throughout. The ml. of the 1.2 N HClused to make up the acid sucrose solution should be reproduced within:0.01 ml. These precautions are necessary if one desires to have theamount of the 0.03 M bicarbonate solutions required to bring the pHbetween 4.0 and 4.8 remain constant within :5 ml. once the proper amountrequired to bring the pH to 4.4 has been determined for the stocksolutions used that day.

A 10 ml. transfer pipette was satisfactory for measuring andtransferring with care the 1.2 N HCl. A 500 ml. graduate sufiiced formeasuring and transferring the 0.03 M bicarbonate solution. The watercould be measured closely enough by noting the volume indicated by therough scale on the sterilization bottle.

The pH values were measured to $0.1 unit by means of pH paper and checkswere made with a pH meter. The small samples of the solutions requiredfor the pH measurement were discarded. In most cases, the pH of eachsolution was determined within i0.1 prior to the sterilization, and thepH values were found to be 4.4:03, The pH increased about 0.1 during thesterilization by heat.

A steam bath or pail partly filled with rapidly boiling water was usedto bring the sucrose solution rapidly to 801-2" C. The temperature ofthe solution was followed, while it was being brought to 80 C. This wasdone by inserting a suitable thermometer into the solution via the holein the rubber bushing which capped the sterilization bottle.

A circulating water bath at 80i1 C. sufiiced for inverting the sucrose.The sucrose solutions were not stirred when they were in the bath ifthey had been preheated to 80 C.

The sugar solutions were mixed adequately by grasping the neck of thecontainer and swirling until the conagitation. The temperature of thesolution is maintained tents rotated rapidly, then the motion wassuddenly re versed and the operation repeated three or four times. Itwas necessary to invert the container several times in order to mix inthe material adhering to the inside of the neck of the bottle; Thecontainer was capped and sealed with stainless steel stoppers duringthese operations unless otherwise noted.

EXAMPLE 5 An approximately solution of invert sugar is prepared in thefollowing manner:

2000 liters of distilled water, equivalent to approximatelythree-fourths of the final voltune of solution desired, is placed in aglass lined tank. The pH of the water-is adjusted with 800 ml. ofconcentrated reagent grade hydrochloric acid to obtain a pH of about2.35, and about 60 grams of sodium bicarbonate are added to remove anamount of oxygen suflicient to prevent the development of color. The pHof the resulting solution is about 2.4. The solution is brought to aboil and 1600 pounds of sucrose are immediately added with constant atthe boiling point for a predetermined period of time necessary for thesubstantially complete inversion of sucrose to invert sugar. The timerequired for this inversion is 30 to minutes.

The pH of the approximately 30% invert sugar solution is adjusted toabout 4.3 by the addition of sodium bicarbonate in the form of a 0.03 Msolution. The percentage of invert sugar in the solution is thendetermined. The solution is diluted to a concentration of 10% invertsugar. The solution is filtered and filled into bottles. A stopper-airtube assembly is placed on each bottle, a partial vacuum created and theremainder of the closure is applied. The pH of the diluted solutionbefore sterilization is 4.6 and after sterilization, it is about 4.2.

. The sealed bottles are sterilized in a pressure autoclave for a periodof 35 minutes at 7 pounds pressure of steam. The sterilized solutionsare allowed to cool.

The colorless solutions prepared in accordance with Examples 1, 2, 3, 4and 5 were found to be substantially free of S-hydroxymethylfurfuralwhen tested in accordance with the method described in Physical andChemical Methods of Sugar Analysis by Browne and Zerban, p. 714,published by John Wiley and Sons, Inc. of N. Y., 1941. Analyses by meansof the polarograph showed that they contained lessS-hydroxymethylfurfural than 0.1% by weight of the hexoses present inthe solutions.

The terms and expressions which I have employed are used as terms ofdescription and not of limitation, and I have no intention, in the useof such terms and expressions, of excluding any equivalents of thefeatures described or portions thereof, but recognize that variousmodifications are possible within the scope of the invention claimed.

What is claimed is:

1. The process of producing a sterile water white solution of invertsugar which contains an amount by weight of S-hydroxymethylfurfural lessthan 0.2% of the hexose present, said process comprising hydrolyzing 98to 99.9% of sucrose in an aqueous solution having a pH of 0.0 to 4.0 inthe substantial absence of an oxidizing agent and at a temperaturewithin the range of 0 to C., and sterilizing by heat said solution undersuch conditions that the sterilized solution has a pH within the rangeof 2.5 to 4.9, the concentration of said sucrose prior to hydrolysisbeing not more than 30% when the hydrolysis is effected at a pH above3.0 and being not more than 10% when the hydrolysis is effected at a pHabove 3.5.

2. The process of producing a sterile water white solution of invertsugar in accordance with claim 1, in which the hydrolysis is effected byhydrochloric acid.

3. The process of producing a sterile water white solution of invertsugar which contains an amount by weight of S-hydroxyrnethylfurfuralless than 0.2% of the hexose present, said process comprisinghydrolyzing 98 to 99.9% of sucrose in an aqueous solution having a pH of0.0 to 3.0 in the substantial absence of an oxidizing agent and at atemperature within the range of to 125 C., and sterilizing by heat saidsolution under such conditions that the sterilized solution has a pHwithin the range of 2.5 to 4.9

4. The process of producing a sterile water white solution of invertsugar which contains an amount by weight of -hydroxymethylfurfural lessthan 0.2% of the hexose present, said process comprising hydrolyzing 98to 99.9% of sucrose in an aqueous solution having a pH of 0.0 to 3.5 inthe substantial absence of an oxidizing agent and at a temperaturewithin the range of 0 to 125 C., and sterilizing by heat said solutionunder such conditions that the sterilized solution has a pH within therange of 2.5 to 4.9, said aqueous solution prior to hydrolysiscontaining not more than 30% of sucrose by weight.

5. The process of producing a sterile water white solution of invertsugar which contains an amount by weight of S-hydroxymethylfurfural lessthan 0.2% of the hexose present, said process comprising hydrolyzing 98to 99.9% of sucrose in an aqueous solution with a non-oxidizing acid ata pH within the range of 0.0 to 3.0 and at a temperature within therange of 0 to 125 C., said aqueous solution being initially treated whenthe hydrolysis is effected at a pH above 2.3 by adding a nontoxic saltof carbonic acid until the pH of the solution is between 2.3 and 3.0,and sterilizing by heat the resulting solution under such conditionsthat the sterilized solution has a pH within the range of 2.5 to 4.9.

6. The process of producing a sterile water white solution of invertsugar which contains an amount by Weight of 5-hydroxymethylfurfural lessthan 0.2% of the hexose present, said process comprising adding sucroseto water having a pH within the range of 0.0 to 3.0, and when the pH isabove 2.3, having the major portion of oxygen removed therefrom,hydrolyzing 98 to 99.9% of said sucrose in the substantial absence of anoxidizing agent and at a temperature within the range of 0 to 125 C.,adjusting the pH of the hydrolyzed solution to a value such that saidhydrolyzed solution after sterilization has a pH within the range of 2.5to 4.9, and sterilizing by heat the resulting hydrolyzed solution.

7. The process of producing a water white solution of invert sugar inaccordance with claim 6, in which the nonoxidizing acid is hydrochloricacid.

8. The process of producing a sterile water white solution of invertsugar which contains an amount by weight of 5-hydroxymethylfurfural lessthan 0.2% of the hexose present, said process comprising adjusting thepH of water to within the range of 2.3 to 3.0 with hydrochloric acid,adding suflicient sodium bicarbonate to remove a major portion of theoxygen from said water and maintaining the pH at not greater than 3.0,adding sucrose to the resulting solution, hydrolyzing 98 to 99.9% ofsaid sucrose at a temperature within the range of 0 to 125 C., adjustingthe pH of the hydrolyzed solution to a value such that said hydrolyzedsolution after sterilization has a pH within the range of 2.5 to 4.9,and sterilizing by heat the resulting adjusted hydrolyzed solution.

9. The process of producing a sterile water white solution of invertsugar which contains an amount by weight of 5-hydroxymethylfurfural lessthan 0.2% of the hexosc present. said process comprising adjusting thepH of water to about 2.3 with hydrochloric acid, adding sufficientsodium bicarbonate to increase the pH to about 2.4, bringing theresulting adjusted solution to a boil at atmospheric pressure, addingsufficient sucrose to produce an aqueous solution of about sucrose,boiling the resulting solution for a period of 30 to minutes tohydrolyze 98 to 99.9% of the sucrose, adjusting the pH of the solutionof invert sugar to about 4.3 with sodium bicarbonate, diluting theadjusted solution with water to a content of about 10% of invert sugar,filling the diluted solution into containers and sterilizing by heat thediluted solution in said containers.

References Cited in the file of this patent UNITED STATES PATENTS1,949,657 Preston Mar. 6, 1934 2,328,191 Cantor Aug. 31, 1943 2,461,163La Lande Feb. 8, 1949 2,534,694 Blann Dec. 19, 1950 2,594,440 HughesApr. 29, 1952 OTHER REFERENCES Bruhns: Int. Sugar Jour., February 1946,pages 51 and 52.

Sugar Analysis, Browne and Zcrban, 3d edition, New York, 1945, pages 405to 415.

Cane Sugar Handbook, Spencer-Meade, 8th edition, New York, 1945, pages676 and 677.

Hawaiian Planters Record, 2nd quarter, 1943, volume 47, Number 2, pages97 to 102 and 104 to 107.

Archiv for Pharmaci og, June 11, 1946, pages 371-374.

1. THE PROCESS OF PRODUCING A STERILE WATER WHITE SOLUTION OF INVERT SUGAR WHICH CONTAINS AN AMOUNT BY WEIGHT OF 5-HYDROXYMETHYLFURFURAL LESS THAN 0.2% OF THE HEXOSE PRESENT, SAID PROCESS COMPRISING HYDROLYZING 98 TO 99.9% OF SUCROSE IN AN AQUEOUS SOLUTION HAVING PH OF 0.0 TO 4.0 IN THE SUBSTANTIAL ABSENCE OF AN OXIDIZING AGENT AND AT A TEMPERATURE WITHIN THE RANGE OF 0* TO 125* C., AND STERILIZING BY HEAT SAID SOLUTION UNDER SUCH CONDITIONS THAT THE STERILIZED SOLUTION HAS A PH WITHIN RANGE OF 2.5 TO 4.9, THE CONCENTRATION OF SAID SUCROSE PRIOR TO HYDROLYSIS BEING NOT MORE THAN 30% WHEN THE HYDROLYSIS IS EFFECTED AT A PH ABOVE 3.0 AND BEING NOT MORE THAN 10% WHEN THE HYDROLYSIS IS EFFECTED AT A PH ABOVE 3.5. 